2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine

Endpoint summary

Description of key information

The skin sensitisation potential of the test substance is assessed in a weight of evidence approach based on three in vitro/in chemico test methods addressing different key events of the skin sensitization AOP, the in silico predictions from the OECD QSAT toolbox and the available LLNA study.

- The test item is likely not to be a skin sensitiser as based on a negative in vitro DPRA prediction assay, performed according to OECD Guideline 442C (K1; Fleet, 2020).

- The negative results of an in vitro KeratinoSens™ assay (ARENrf2 Luciferase Test) performed according to OECD Guideline 442D (K1; Kelsall, 2019), are supporting the prediction that the test item is not a skin sensitizer.

- The test item showed a positive result in an in vitro human Cell Line Activation Test (h-CLAT) method, performed according to OECD Guideline 442E (K1; Jacobs, 2020).

A prediction for in silico alerts for protein binding and the automated workflow for skin sensitisation was made in the OECD QSAR Toolbox which predict the test item as negative for protein binding and for sensitisation. 

The Local Lymph Node Assay performed according to OECD Guideline 429, a SI exceeding 3 was observed at 2.5% and 5%, however the ear thickness increase was above 25% which indicated excessive irritation (Klatt, 2015). 

In a weight-of-evidence approach, all available data is considered, and it was concluded that it does not warrant the classification as skin sensitiser. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

6 October 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox 4.4.1
2. MODEL (incl. version number)
Automated workflow for Skin sensitization for defined
approaches

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CN(C)CCOCCN(C)CCOCCN
(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: see attached documentation
- Unambiguous algorithm: see attached documentation
- Defined domain of applicability: see attached documentation
- Appropriate measures of goodness-of-fit and robustness and predictivity: see attached documentation
- Mechanistic interpretation: see attached documentation

5. APPLICABILITY DOMAIN
The Automated workflow for Skin sensitization for defined approaches of the OECD QSAR toolbox 4.4.1 automatically verifies if the input is within the applicability domain of the model.
The input structure was withing the applicability domain.

6. ADEQUACY OF THE RESULT
The prediction is adequate to be used as part of a defined approach for skin sensitisation GD 497.

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox 4.4.1.
- Model(s) used: Automated workflow for Skin sensitization for defined approaches
- Model description: see attached documents
- Justification of QSAR prediction: the prediction is adequate for use as part of a defined approach for skin sensitisation prefiction (GD 497)

Parameter:

other: negative

Value:

0

Interpretation of results:

other: Negative

Conclusions:

The OECD QSAR Toolbox Automated workflow for Skin sensitization for defined
approaches predicts the test item to be negative or skin sensitisation. No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2020-01-27 to 2020-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test {h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis

Version / remarks:

23 July 2018

Deviations:

no

Principles of method if other than guideline:

Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
The h-CLAR has been evaluated in an European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: PFW190073
- Expiry date: 2021-02-05
- Physical State: Clear colourless liquid
- Purity: 99.30%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (10-30°C)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

CELL CULTURE:
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0L Y, UK. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived.

SOLUBILITY ASSESSMENT:
Solubility was first determined for the test item using cell culture medium (RPMI 1640). Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid.

DOSE RANGE FINDING (CELL VIABILITY):
- CV75 testing followed (2 independent runs) where THP-1 cells were pre-cultured for 72hrs then dosed with the test item over an 8-dose range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide and the discrimination of live/dead cells was measured by flow cytometry. The dose of test item that yielded 75% cell viability (CV75) was calculated and used to create a narrower test item dilution range for the next stage of testing.
- Concentrations tested: 5000, 2500, 1250, 625, 312.50, 156.25, 78.13 and 39.06 μg/ml

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- CD54 and CD86 marker expression was then performed (four independent runs). THP-1 cells were pre-cultured for 72hrs then dosed with the test item over the narrower dilution range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide, antibodies that detect CD54 and CD86 marker expression and a negative control antibody. The discrimination of live/dead cells and changes in CD54 and CD86 marker expression were measured by flow cytometry.
- Data was only collected for two independent runs due to two runs being postponed prior to result data generation and collection. These were postponed due to consumable supply issue. Any written documentation generated prior to the postponement of the two runs is retained in the study file.
- Concentrations tested:2488.64, 2073.87, 1728.23, 1440.19, 1200.16, 1000.13, 833.44 and 694.53 μg/ml

DATA EVALUATION
- Cytotoxicity assessment
- Prediction model used

CV75 ACCEPTANCE CRITERIA:
- Cell viability must be 2: 75% at the lowest dose.
- The highest test item concentration should produce cytotoxicity (< 90% cell viability) unless 5mg/ml in medium, 1 mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item.

CD54/CD86 EXPRESSION: RUN ACCEPTANCE CRITERIA:
- Cell viability of medium and DMSO controls should be greater than 90%
- In the positive control (Nickel Sulphate; 100μg/ml), RFI values of both CD86 and CD54 should meet the criteria for a positive result (CD86 2: 150 and CD54 2: 200) and cell viability should be > 50%
- In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not cross the threshold that denotes a positive response (CD86 2: 150 and CD54 2: 200)
- For both medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

CD54/CD86 EXPRESSION: DATA ACCEPTANCE CRITERIA:
- For a test item resulting in a negative value (i.e. Non-Sensitiser), the cell viability at the 1.2 x CV75 value should be less than 90%
- For a test item resulting in a positive value (i.e. Sensitiser), a cell viability at the 1.2 x CV75 value of more than 90% is considered acceptable
- When test items in RPMI are tested at 5mg/ml or test items in DMSO are tested at 1 mg/ml or the highest soluble concentration is used as the maximal dose instead of the CV75 based dose, the data for the test item are accepted regardless of the cell viability
- Cell viability of at least 4 doses in each assay should be > 50%.

ABNORMAL VALUES:
- RFI values cannot be less than zero. Such values should be excluded from the prediction.
- If an abnormal value such as strongly induced CD54 or CD86 expression at only one noncytotoxic concentration is observed, this should be checked to determine if there were any abnormal events/conditions in the run and the details recorded.

Vehicle / solvent control:

cell culture medium

Negative control:

not applicable

Positive control:

other: 2,4-dinitrochlorobenzene (DNCB) for CV75 and Nickel Sulphate for CD54 and CD86 Expression

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

1 033 µg/mL

Cell viability:

> 50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

919 µg/mL

Cell viability:

> 50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

1 103 µg/mL

Cell viability:

> 50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Value:

1 207 µg/mL

Cell viability:

> 50%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

SOLVENT SELECTION AND CV75 DETERMINATION:
- Prior to the CV75 determination, the test item was assessed for solubility and was found to be soluble in RPMI (Culture Medium) at 500 mg/ml.
- The CV75 value derived from two independent experiments was as follows: Run 1: 2500 μg/ml and Run 2: 1647.73 μg/ml (average: 2073.87 μg/ml)

ACCEPTANCE OF RESULTS:
Acceptance criteria for all controls and the test item were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.
- CV75 Acceptance criteria:
*Cell viability must be >=75% at the lowest dose.
*The highest test item concentration should produce cytotoxicity (< 90% cell viability) unless 5mg/ml in medium, 1 mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item.
- CD54/CD86 Expression: Run and data acceptance criteria:
* Cell viabilities of medium and solvent Medium controls should be higher than 90%.
*In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI >=150% and
CD54 RFI >=200%) compared to the medium control.
*For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
*In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI >=150 and CD54 RFI >=200) and cell viabilitv should be greater than 50%.
*For each test item, the cell viability should be greater than 50% in at least four tested concentrations in each run.
*Negative results are acceptable only for test items exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/ml in medium, 1 mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item.

DISCUSSION:
- The CV75 assay provided the narrower test item dilution range for the CD54/86 expression assay.
- The top dose for the CD54/86 expression assay (main test) was 1.2 x CV75 Value which was equal to 2488.64μg/ml.
- the expression of CD54 as measured by the RFI crossed the threshold (RFI ~200) at all concentrations greater than and including 1200.16μg/ml for Run 3, and at all concentrations greater than and including 1440.19 μg/ml for Run 4.
- The expression of CD86 as measured by the RFI crossed the threshold (RFI ~150) at all concentrations greater than and including 1200.16μg/ml for Run 3, and at all concentrations greater than and including 1000.13 μg/ml for Run 4.
- As the CD54/CD86 expression crossed the threshold the test item is classified as Positive.
- Cell viability did not fall below 50% at any of the test item concentrations and therefore the result is deemed to be valid.

Interpretation of results:

other: Activated THP-1 cells

Conclusions:

For the test substance, the dose that gave 75% cell viability the highest soluble concentration was found to be 2073.87 μg/ml. The EC200 and EC150 values for CD54 and CD86 expression were 1207 and 1033 respectively. Therefore, the test substance was predicted positive as per the prediction model. No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-08-27 to 2019-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

yes

Remarks:

See section 'Any other information on material & methods'

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol no 155: KeratinoSens™

Version / remarks:

March 2018

Deviations:

yes

Remarks:

See section 'Any other information on material & methods'

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Lot/batch number of test material: PFW190073
- Appearance: Clear liquid
- Expiry/retest date: 2021-02-05
- Purity: 99.3%
- molecular weight: 261.40 g/mol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (10 - 30˚C), in the dark
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Prior to commencing testing, the solubility of the test item in assay medium containing 1% DMSO at 200 mM was assessed. The test item, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

CELL CULTURE:
- The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10500) and 5.5 mL Geneticin (Gibco 10131).
- Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196°C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in 9 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.


PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS:
- Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.
- The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 μL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 μL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

PREPARATION OF THE POSITIVE CONTROL:
Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the following formula:

V= (p÷100)×W/MW − W/1000

Where
V = volume of DMSO in mL to be added p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg

The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO. Preparation of the positive control was shared with other studies performed in the same assay.


PREPARATION OF THE SOLVENT CONTROL:
- well in column 12. 200 μL of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11 as per the example plate layout on page 12. One well was left blank. All other wells contained 100 μL of the appropriate solvent. Serial halving dilutions of the test item were prepared by transferring 100 μL from each dilution into 100 μL of solvent. Pipette tips were discarded after each transfer and then fresh pipette tips were used to mix each concentration prior to the next transfer. The 6.4 mM stock solution of cinnamic aldehyde was diluted from column 11 to column 7 using the same procedure of serial halving dilutions.
- The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 μL of assay medium to each well and then 10 μL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

TEST SYSTEM:
A stock solution of the test item, JEFFCAT® LE-30, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

EXPERIMENTAL DESIGN:
- Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.
- The second test was conducted between 23 September 2019 and 26 September 2019, however, the results for the average coefficient of variation of the luminescence reading for the DMSO solvent control was above the acceptance criterion of below 20%. The test was repeated between 30 September 2019 and 03 October 2019. Only the data and results from test 1 and the repeat of test 2 are included in this report.
- Treatment of Cultured Plates:
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 μL of assay medium was added to every well of the 96 well plates. 50 μL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.
- Cell Viability Measurement:
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 μL fresh assay medium was added to each well. 10 μL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 μL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.
- Luciferase Measurements:
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for the test 2. Frozen reconstituted reagent was used for test 1 and was thawed to room temperature before use. After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 μL of fresh assay medium was added to each well before 100 μL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

DATA EVALUATION:
The following parameters were calculated in the KeratinoSens™ test method:
- the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
- the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
- the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

ACCEPTABILITY CRITERIA:
In order for an assay to be accepted the following criteria must be met:
- The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using a t-test) in at least one of the tested concentrations (4 to 64 μM).
- The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
- The average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

DATA INTERPRETATION:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is ≥1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is >70% at the lowest concentration with induction of luciferase activity ≥1.5 fold (i.e. at the EC1.5 determining concentration);
- the EC1.5 value is < 1000 μM;
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).
If in a given test, all of the first three conditions listed above are met, but a clear doseresponse for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 μM and that do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was
statistically significant above the threshold of 1.5 in at least one of the tested concentrations
(4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 6.34 μM and 6.31 μM for
test 1 and 2, respectively, which lay within the historical control range for this laboratory . The average induction in the three replicates for cinnamic aldehyde at 64 μM were
4.64 and 14.23 for test 1 and 2, respectively. The result for test 1 met the acceptance criterion
of between 2 and 8. However, the result for test 2 did not meet

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

0.93

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

1 373.92 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

658.95 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

1 124.6 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

511.05 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no relevant increase

Remarks:

no EC1.5 value calculated

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no relevant increase

Remarks:

no EC1.5 value calculated

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

- The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 10.8% and 19.9% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Interpretation of results:

other: Does not activate keratinocytes

Conclusions:

It was concluded that the test item gave a negative response in the ARENrf2 Luciferase Test (KeratinoSens™). No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-09-13 to 2019-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Lot/batch number of test material: PFW190073
- Appearance: Clear liquid
- Expiry/retest date: 2021-02-05
- Purity: 99.3%
- Molecular weight: 261.4 g/mol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, 15°C to 25°C
- solubility of the test substance: The solubulity of the test substance was assessed at a concentration of 100 mM in a selection of solvents

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

- Preparation of the test chemical solutions: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

- Preparation of the positive controls, reference controls and co-elution controls:
* Preparation of Stability Controls and Precision Controls: Stability controls (Reference Control B, n=6, Reference Control C, n=3) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM. Reference Control A and Reference Control B were prepared with buffer and acetonitrile, Reference Control C was prepared with acetone.
* Preparation of Positive Control Solution and Test Item Stock Solution: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of the test substance was prepared in acetone.
* Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Triplicate solutions each of the test substance and the positive control stocks were diluted with the Cysteine peptide to prepare final solutions containing 0.5 mM Cysteine and 5 mM of either the test substance or the positive control. For the co-elution control, blank buffer solution was used in place of the Cysteine stock solution.
* Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls Triplicate solutions each of the test substance and the positive control stocks were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test substance or the positive control. For the co-elution control, blank buffer solution was used in place of the Lysine stock solution.

INCUBATION
- Incubation conditions: The appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
- Verification of the suitability of the HPLC for test chemical and control substances
- The concentration of each peptide in the presence of the test substance and the associated positive controls were quantified by HPLC using UV detection as detailed in the chromatographic section.

DATA EVALUATION
- Cys and Lys peptide detection wavelength

Vehicle / solvent:

acetone

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

-2.35 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

-0.289 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

No co-elution occurred during either the cysteine or the lysine assay.

Solubility assessment: the solubility of the test substance in acetone at a nominal concentration of 100 mM was confirmed. The test substance was insoluble in acetonitrile at 100 mM.

Interpretation of results:

other: Non peptide binding

Conclusions:

Solutions of the test substance were successfully analysed by the validated DPRA analytical method (Covance Analytical Method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With no overall peptide depletion, the DPRA prediction for the test substance is therefore negative. However, no predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-06-26 to 2014-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004/73/EC L152

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: PFW120356
- Expiration date of the lot/batch: Not declared
- Purity: 98.70%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20-23°C)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, Frederick, MD
- Age at study initiation:
Preliminary Irritation Screen - 7-8 weeks
Main Study - 9 weeks
- Weight at study initiation:
Preliminary Irritation Screen - 18-25 grams
Main Study - 20 to 24 grams
- Housing: Animals were group-housed 5 per cage upon receipt and during study in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals".
- Diet (e.g. ad libitum): free access to Harlan Teklad Rodent Diet
- Water (e.g. ad libitum): ad libitum, via water bottles.
- Acclimation period: at least 8 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
AeroNeg 1 (9- 22 Jul 2014): 24-27°C
AeroNeg 2 (22 Jul - 11 Aug 2014): 19-27°C
- Humidity (%):
AeroNeg 1 (9-22 Jul 2014): 24-98%
AeroNeg 2 (22 Jul - 11 Aug 2014): 24-97%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary study (irritation screen): 25%, 50% and 100% (v/v)
Second irritation screen: 2.5, 5 and 10% (v/v)
Main test: 1, 2.5 and 5% v/v

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
Prior to the main study, an irritation screen was performed using the substance at 25, 50 and 100% (v/v). Each concentration was applied to the dorsal surface of both ears of 2 mice once per day for 3 consecutive days. Mice were observed daily for any clinical signs of systemic toxicity. Body weights were recorded pre-test and on Day 6. Ears of each mouse were observed for erythema and scored daily using the Draize scoring system. Ear thickness measurements were taken on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Because of observed toxicity and excessive local irritation, a second irritation screen was run using the substance at 2.5, 5 and 10% (v/v).
- Irritation: excessive local irritation in the first irritation screen, therefore a second irritation screen was run using the substance at lower concentrations
- Systemic toxicity: observed in first irritation screen, therefore a second irritation screen was run using the substance at lower concentrations; in the second irritation screen, all animals were clinically normal throughout the study.
- Ear thickness measurements: The appearance of the ears, which were the application sites, appeared hard and reduced in size on Day 6 in the group treated with 25%. The ears in the group treated with 25% could not be measured on Day 6. Therefore, a second irritation screen was run with the substance at concentrations of 2.5, 5 and 10% (v/v). In the second irritation screen, the ears in the group treated with the test article at 10% had a hard feel to them on Day 6. The ear measurements indicated little or no edema in the ears of the mice treated with the test article at 2.5 and 5%, but treatment with 10% caused a 22.7% increase in ear thickness on day 3 and a 67.5% increase on Day 6, indicating the presence of edema.
- Erythema scores: first irritation screen: ; second irritation screen: There was very slight erythema on the ears of the mice treated with the test article at 2.5 and 5%. Very slight to well-defined erythema was seen on the ears of the mice treated with 10%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The criterion for a positive response was that one or more concentrations of a test article elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION: The test article formulations will be prepared on each day of dosing by dissolving the appropriate amount of test article in the vehicle using a volumetric flask for each concentration. The positive control will be prepared as a 35% solution in the vehicle.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean DPM and standard error (sem) for each group was determined.
Increases in ³H-thymidine incorporation relative to the vehicle-treated control was derived for each group and recorded as stimulation indeces (SI).
Body weight, body weight changes and ear measurements were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc. The evaluation of equality of means for body weight data and ear measurements was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the log DPM was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
The percent change in ear thickness from Day 1 was determined for both ears of each mouse on Days 3 and 6. The mean percent change for each group was calculated.
The EC3 was calculated using the formula:
EC3 = c+[(3-d)/(b-d)](a-c)
where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/J mice. The validation- /positive control study was performed with alpha-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v).
The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 10.5. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

1%

Key result

Parameter:

SI

Value:

6.9

Test group / Remarks:

2.5%

Remarks on result:

other: ear thickness increase exceeded 25%

Key result

Parameter:

SI

Value:

5.1

Test group / Remarks:

5%

Remarks on result:

other: ear thickness increase exceeded 25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM (mean ± sem):
Vehicle: 1207.51 ± 55.0
1% v/v: 2704.66 ± 364.2
2.5% v/v: 8328.50 ± 55.6
5% v/v: 6204.23 ± 549.3

DETAILS ON STIMULATION INDEX CALCULATION
Concentrations of 1.0, 2.5, and 5% resulted in SI of 2.2, 6.9, and 5.1, respectively, when compared to the vehicle. There was a statistically significant difference between the groups treated with the test article at 1 % (p<0.05) and 2.5% and 5% (p < 0.001) when compared to the vehicle control group. These differences were biologically relevant since the SIs were> 3.0

EC3 CALCULATION
EC3 = 1.26%

CLINICAL OBSERVATIONS:
In the main study, there was no mortality and all animals appeared clinically normal. The ears of the mice treated with HCA appeared wet on Days 2-6. The ears of the mice treated with test item at 5% appeared wet on Days 2-5 while the ears of the mice treated with test item at 1 % or 2.5% appeared wet on days 3-5. There were no other findings.

DERMAL IRRITATION
There was no visible erythema at the application site of any of the animals in the substance 1% or 2.5% group or the vehicle treated groups. All animals treated with the substance at 5% or HCA had very slight erythema on Days 3-6.
There was also no edema at the application site of any of the mice treated with the test article at 1% or vehicle. Mean increases in ear thickness for groups treated with the substance at 1% or vehicle were < 25% on Days 3 and 6. Edema was observed in the ears of the mice treated with the substance at 2.5 and 5% and HCA. Mean increases in ear thickness for the substance at 2.5% treated group were 12.4% on Day 3 and 26.9% on Day 6. These increases in ear thickness for the substance at 5% treated group were 31.5% on Day 3 and 38.0% on Day 6 which were statistically significant (p<0.01). Mean increases in ear thickness for the HCA treated group were 41.4% on Day 3 and 58.6% on Day 6 which were statistically significant (p<0.001).

BODY WEIGHTS
In the initial irritation screen there were no significant changes in body weights in any of the animals.
In the second irritation screen there were no significant changes in body weights in any of the animals
In the main study there were no statistically significant differences. Therefore, the test article did not appear to cause any overt toxicity at 1, 2.5 or 5%.

Interpretation of results:

GHS criteria not met

Conclusions:

In the study, treatment with the substance at 2.5, and 5% did result in a stimulation index greater than 3.0. However, treatment with the substance at 2.5 and 5% induced an increase of ear thickness greater than 25% which shows excessive irritation. At 1% the SI was 2.2 and the ear thickness increase was below 25% which fulfills the validity criteria for the assay. Based on this result the GHS criteria are not met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential of the test substance was assessed in a weight-of-evidence approach, addressing all available information (see attached document for further details). The following information was included: physico-chemical properties of the substance, the results of in silico screening for potention alerts, a combination of several methods addressing key events of the adverse outcome pathway for skin sensitisation as defined by OECD 497 and the LLNA. 

The test substance has been tested in 3 different non-animal methods addressing different key events of the skin sensitization AOP which resulted in one positive result in the h-CLAT and two negative results in DPRA and KeratinoSensTM. The dataset was complemented with the prediction for in silico alerts for protein binding and the read across produced with the OECD QSAR toolbox automated workflow for skin sensitization.

A single in vitro assay is not sufficient to determine the skin sensitization hazard (or potency) of a substance. For this reason, the in vitro assays in this report were evaluated based on the "2 out of 3" integrated testing strategy approach to skin hazard identification and the ITS v2 (OECD 497) because the available data could be used for both defined approaches. For the “2 out of 3” DA the prediction is made based on the majority call of any 2 assays. The combination of two negative results (DPRA and KeratinoSens^TM) and one positive result (h-CLAT) gives a prediction as not a skin sensitiser for the test item.

In the ITS v2 the prediction is made by combining the scores obtained with the results of the DPRA (depletion 0%, score 0), the Minimum induction threshold of the hCLAT assay (MIT= 919 μg/mL; score 1) and the hazard prediction for sensitization by the automated workflow of the OECD QSAT TB (negative prediction; score 0). Based on the prediction model of the defined approach the total score of 1 means that the substance is not -classified for skin sensitization.

The substance has been tested in the LLNA assay where it produced a positive result at 2.5% and 5% with an EC3 value of 1.26%. However, the irritation measured at those two concentrations was above the 25% cut-off that is recommended by the test guideline as there is a tendency for irritants to cause false positive results in the LLNA. 

Given the fact that the test substance does not have any structural alerts for skin sensitization, that in vitro it gave a negative result in the defined approach "2 out of 3" and also in the ITS v2 but it was tested positive in the LLNA only at concentrations that showed excessive ear swelling we concluded that it does not warrant the classification as skin sensitiser. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results, the test substance does not trigger protein binding alerts, is negative in the OECD QSAR toolbox automated workflow based on read-across from analogues, is not peptide reactive, does not activate keratinocytes and activated dendritic cells at high concentrations. It shows a positive result in an LLNA assay only at concentrations which cause excessive ear swelling. The weight of evidence integration of the available information suggests that the test substance should not be classified for skin sensitisation.